Part:BBa_K634007:Experience

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Applications of BBa_K634007

When placed 3' of the promoter portion of a 1- or 2-component sensor, BBa_634007 can be used in the following ways:

Reporter

tagRFP (BBa_K634005) fluoresces red upon induction. This can be useful for visually verifying expression of the cassette is proceeding normally, first order approximations of promoter activity, and (with plate reader analysis) as a marker for quantification of expression. Cell cultures have not appeared visibly red in our experience, but are easy to see when pelleted (see example).

Selection

Sensor systems can be selected for desired induced expression through supplementation of the culture with the sensor's analyte and kanamycin.

Counter selection

Sensors can also be selected for absence of expression when uninduced through supplementation of the culture with sucrose

Issues in practice

sacB appears to be something of a lightning rod for loss of function mutations. As can be seen below, sucrose lethality is only maintained in a population for approximately 5 hours. At this point, we presume that spontaneous mutations in sacB produce sucrose-insensitive mutants.

User Reviews

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